Anterior cruciate ligament xenografts

ABSTRACT

The invention provides an article of manufacture comprising a substantially non-immunogenic ligament or tendon xenograft for implantation into humans. The invention further provides a method for preparing a ligament xenograft by removing at least a portion of a ligament from a non-human animal to provide a xenograft; washing the xenograft in saline and alcohol; subjecting the xenograft to at least one treatment selected from the group consisting of exposure to ultraviolet radiation, immersion in alcohol, ozonation, freeze/thaw cycling, and optionally chemical crosslinking. In addition to or in lieu of the above treatments, the methods include a cellular disruption treatment and either digestion of the carbohydrate moieties of the xenograft with a glycosidase in a range of about 1 mU/ml to about 1000 U/ml or glycosidase digestion followed by treatment for sialylation. The invention also provides articles of manufacture produced by one or more of the above-identified methods of the invention. The invention further provides a ligament xenograft for implantation into a human including a portion of a ligament from a non-human animal, wherein the portion includes extracellular components and substantially only dead cells having substantially no surface -galactosyl moieties and having sialic acid linked to at least a portion of surface carbohydrate moieties. Each of the xenografts of the invention is substantially non-immunogenic and has substantially the same mechanical properties as the respective native ligament.

RELATED APPLICATION

[0001] This application is a continuation of U.S. Ser. No. 09/824,327,filed Apr. 2, 2001, which is a continuation of U.S. Ser. No. 08/529,199,filed Mar. 6, 1998, now U.S. Pat. No. 6,210,440, which is acontinuation-in-part of U.S. Ser. No. 08/529,199, filed Dec. 1, 1997,now U.S. Pat. No. 5,902,338, which is a Continued ProsecutionApplication (CPA) of U.S.Ser. No. 08/529,199, filed Sep. 15, 1995.

FIELD OF THE INVENTION

[0002] The present invention relates to the field of surgical repair ofinjuries of the anterior cruciate ligament in the human knee using asubstantially immunologically compatible ligament or tendon from anon-human animal to replace the damaged human anterior cruciateligament.

BACKGROUND OF THE INVENTION

[0003] The anterior cruciate ligament of the knee (hereinafter the ACL)functions to resist anterior displacement of the tibia from the femur atall flexion positions. The ACL also resists hyperextension andcontributes to rotational stability of the fully extended knee duringinternal and external tibial rotation. The ACL may play a role inproprioception. The ACL is made up of connective tissue structurescomposed of cells, water, collagen, proteoglycans, fibronectin, elastin,and other glycoproteins. Cyril Frank, M.D. et al., Normal Ligament:Structure, Function, and Composition. Injury and Repair of theMusculoskeletal Soft Tissues, 2:45-101. Structurally, the ACL attachesto a depression in the front of the intercondyloid eminence of the tibiaextending postero-superiorly to the medial wall of the lateral femoralcondyle.

[0004] Partial or complete tears of the ACL are very common, comprisingabout 30,000 outpatient procedures in the U.S. each year. The preferredtreatment of the torn ACL is ligament reconstruction, using abone-ligament-bone autograft. Cruciate ligament reconstruction has theadvantage of immediate stability and a potential for immediate vigorousrehabilitation. However, the disadvantages to ACL reconstruction aresignificant: for example, normal anatomy is disrupted when the patellartendon or hamstring tendons are used for the reconstruction; placementof intraarticular hardware is required for ligament fixation; andanterior knee pain frequently occurs. Moreover, recent reviews ofcruciate ligament reconstruction indicate an increased risk ofdegenerative arthritis with intraarticular ACL reconstruction in largegroups of patients.

[0005] A second method of treating ACL injuries, referred to as “primaryrepair”, involves suturing the torn structure back into place. PrimaryACL repair has the potential advantages of a limited arthroscopicapproach, minimal disruption of normal anatomy, and an out-patientprocedure under a local anesthetic. The potential disadvantage ofprimary cruciate ligament repair is the perception that over the longterm ACL repairs do not provide stability in a sufficient number ofpatients, and that subsequent reconstruction may be required at a laterdate. The success rate of anterior cruciate ligament repair hasgenerally hovered in the 60% to 70% range.

[0006] Much of the structure and many of the properties of originaltissues may be retained in transplants through use of xenograft orheterograft materials, that is, tissue from a different species than thegraft recipient. For example, tendons or ligaments from cows or otheranimals are covered with a synthetic mesh and transplanted into aheterologous host in U.S. Pat. No. 4,400,833. Flat tissues such as pigpericardia are also disclosed as being suitable for heterologoustransplantation in U.S. Pat. No. 4,400,833. Bovine peritoneum fabricatedinto a biomaterial suitable for prosthetic heart valves, vasculargrafts, burn and other wound dressings is disclosed in U.S. Pat. No.4,755,593. Bovine, ovine, or porcine blood vessel xenografts aredisclosed in WO 84/03036. However, none of these disclosures describethe use of a xenograft for ACL replacement.

[0007] Once implanted in an individual, a xenograft provokes immunogenicreactions such as chronic and hyperacute rejection of the xenograft. Theterm “chronic rejection”, as used herein, refers to an immunologicalreaction in an individual against a xenograft being implanted into theindividual. Typically, chronic rejection is mediated by the interactionof IgG natural antibodies in the serum of the individual receiving thexenograft and carbohydrate moieties expressed on cells, and/orextracellular components. For example, transplantation of ligament ortendon xenografts from nonprimate mammals (e.g., porcine or bovineorigin) into humans is primarily prevented by the interaction betweenthe IgG natural anti-Gal antibody present in the serum of humans withthe carbohydrate structure Gal 1-3Gal 1-4G1cNAc-R ( -galactosyl or -galepitope) expressed in the xenograft. K. R. Stone et al., Porcine andbovine cartilage transplants in cynomolgus monkey: I. A model forchronic xenograft rejection, 63 Transplantation 640-645 (1997); U.Galili et al., Porcine and bovine cartilage transplants in cynomolgusmonkey: II. Changes in anti-Gal response during chronic rejection, 63Transplantation 646-651 (1997). In chronic rejection, the immune systemtypically responds within one to two weeks of implantation of thexenograft.

[0008] In contrast with “chronic rejection”, “hyper acute rejection” asused herein, refers to the immunological reaction in an individualagainst a xenograft being implanted into the individual, where therejection is typically mediated by the interaction of IgM naturalantibodies in the serum of the individual receiving the xenograft andcarbohydrate moieties expressed on cells. This interaction activates thecomplement system causing lysis of the vascular bed and stoppage ofblood flow in the receiving individual within minutes to two to threehours.

[0009] The term “extracellular components”, as used herein, refers toextracellular water, collagen, proteoglycans, fibronectin, elastin, andother glycoproteins present in the ligament or tendon.

[0010] Xenograft materials may be chemically treated to reduceimmunogenicity prior to implantation into a recipient. For example,glutaraldehyde is used to cross-link or “tan” xenograft tissue in orderto reduce its antigenicity, as described in detail in U.S. Pat. No.4,755,593. Other agents such as aliphatic and aromatic diamine compoundsmay provide additional crosslinking through the side chain carboxylgroups of aspartic and glutamic acid residues of the collagenpolypeptide. Glutaraldehyde and diamine tanning also increases thestability of the xenograft tissue.

[0011] Xenograft tissues may also be subjected to various physicaltreatments in preparation for implantation. For example, U.S. Pat. No.4,755,593 discloses subjecting xenograft tissue to mechanical strain bystretching to produce a thinner and stiffer biomaterial for grafting.Tissue for allograft transplantation is commonly cryopreserved tooptimize cell viability during storage, as disclosed, for example, inU.S. Pat. Nos. 5,071,741; 5,131,850; 5,160,313; and 5,171,660. 5,071,741discloses that freezing tissues causes mechanical injuries to cellstherein because of extracellular or intracellular ice crystal formationand osmotic dehydration.

SUMMARY OF THE INVENTION

[0012] The present invention provides a substantially non-immunogenicligament or tendon xenograft for implantation into a human in need ofACL repair or replacement. The invention further provides methods forprocessing xenogeneic ligaments or tendons with reduced immunogenicitybut with substantially native elasticity and load-bearing capabilitiesfor xenografting into humans.

[0013] As described herein, the term “ligament” also includes tendons.

[0014] As described herein, the term “xenograft” is synonymous with theterm “heterograft” and refers to a graft transferred from an animal ofone species to one of another species. Stedman's Medical Dictionary,Williams & Wilkins, Baltimore, MD (1995).

[0015] As described herein, the term “xenogeneic”, as in xenogeneicgraft ligament, etc., refers to a graft, ligament, etc., transferredfrom an animal of one species to one of another species. Id.

[0016] The methods of the invention, include, alone or in combination,treatment with radiation, one or more cycles of freezing and thawing,treatment with a chemical cross-linking agent, treatment with alcohol orozonation. In addition to or in lieu of these methods, the methods ofthe invention include a cellular disruption treatment and digestion ofthe carbodydrate moieties of the xenograft with a glycosidase in aconcentration range of about 1 mU/ml to about 1000 U/ml or glycosidasedigestion followed by capping of carbohydrate moieties of the xenograftwith sialic acid. After one or more of the above-described processingsteps, the methods of the invention provide a xenograft havingsubstantially the same mechanical properties as a corresponding portionof a native ligament.

[0017] As described herein, the term “cellular disruption” as in, forexample, cellular disruption treatment, refers to a treatment forkilling cells.

[0018] As described herein, the term “capping molecules”, refers tomolecules which link with carbohydrate chains such that the xenograft isno longer recognized as foreign by the subject's immune system.

[0019] In one embodiment, the invention provides an article ofmanufacture comprising a substantially non-immunogenic ligamentxenograft for implantation into a human.

[0020] In another embodiment, the invention provides a method ofpreparing a ligament xenograft for implantation into a human, whichincludes removing at least a portion of a ligament from a non-humananimal to provide a xenograft; washing the xenograft in water andalcohol; and subjecting the xenograft to at least one treatment selectedfrom the group consisting of exposure to ultraviolet radiation,immersion in alcohol, ozonation, and freeze/thaw cycling, whereby thexenograft has substantially the same mechanical properties as acorresponding portion of a native ligament.

[0021] As described herein, the term “portion”, as in, for example, aportion of ligament or a portion of surface carbohydrate moieties,refers to all or less than all of the respective ligament or surfacecarbohydrate moieties.

[0022] In still another embodiment, the invention provides a method ofpreparing a ligament xenograft for implantation into a human, whichincludes removing at least a portion of a ligament from a non-humananimal to provide a xenograft; washing the xenograft in water andalcohol; subjecting the xenograft to a cellular disruption treatment;digesting the xenograft with a glycosidase in a concentration range ofabout 1 mU/ml to about 1000 U/ml to remove substantially first surfacecarbohydrate moieties from the xenograft, whereby the xenograft issubstantially non-immunogenic and has substantially the same mechanicalproperties as a corresponding portion of a native ligament.

[0023] In a further embodiment, the invention provides a method ofpreparing a ligament xenograft for implantation into a human, whichincludes removing at least a portion of a ligament from a non-humananimal to provide a xenograft; washing the xenograft in water andalcohol; subjecting the xenograft to a cellular disruption treatment;digesting the xenograft with a glycosidase to remove substantially firstsurface carbohydrate moieties from the xenograft; and treating secondsurface carbohydrate moieties on the xenograft with sialic acid to capat least a portion of the second surface carbohydrate moieties, wherebythe xenograft is substantially non-immunogenic and has substantially thesame mechanical properties as a corresponding portion of a nativeligament.

[0024] As described herein, the terms “to cap” or “capping”, refer tolinking a carboydrate unit to the end of a carbohydrate chain, as in,for example, covalently linking salic acid to surface carbohydratemoieties on the xenograft.

[0025] In still further embodiments, the invention provides articles ofmanufacture including substantially non-immunogenic ligament xenograftsfor implantation into humans produced by one or more of theabove-identified methods of the invention.

[0026] In yet another embodiment, the invention provides a ligamentxenograft for implantation into a human which includes a portion of aligament from a non-human animal, wherein the portion includes aextracellular components and substantially only dead cells, theextracellular matrix and the dead cells having substantially no surface-galactosyl moieties and having sialic acid molecules linked to at leasta portion of surface carbohydrate moieties. The ligament portion issubstantially non-immunogenic and has substantially the same mechanicalproperties as as a corresponding portion of a native ligament.

BRIEF DESCRIPTION OF THE DRAWING

[0027]FIG. 1 is a graphical representation of the specificity ofmonoclonal anti-Gal antibodies for -galactosyl epitopes on bovine serumalbumin (BSA), bovine thyroglobulin, mouse laminin, Gal 1-4 G1cNAc-BSA(N-acetyllactosamine-BSA), Gal 1-4Gal 1-4G1cNAc-BSA (P1 antigen linkedto BSA), and human thyroglobulin or human laminin.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] The present invention is directed against the chronic rejectionof xenografts for implantation into humans. Accordingly, the ligamentxenograft produced in accordance with the method of the invention issubstantially non-immunogenic, while generally maintaining themechanical properties of a native ligament. While the ligament mayundergo some shrinkage during processing, a ligament xenograft preparedin accordance with the invention will have the general appearance of anative ligament. The ligament xenograft may also be cut into segments,each of which may be implanted into the knee of a recipient as set forthbelow.

[0029] The invention provides, in one embodiment, a method for preparingor processing a xenogeneic ligament for engraftment into humans. Theligament may be harvested from any non-human animal to prepare thexenograft of the invention. Ligaments from transgenic non-human animalsor from genetically altered non-human animals may also be used asxenografts in accordance with the present invention. Preferably, bovinejoints serve as sources of the ligament used to prepare the xenografts.More preferably, immature joints from immature animals are the sourcesof the ligament, since the tissue of younger animals may be inherentlymore elastic and engraftable than that of older animals. Mostpreferably, the age of the source animal is between six and eighteenmonths at time of slaughter. Additionally, the patellar tendon, theanterior or posterior cruciate ligaments, the Achilles tendon, or thehamstring tendons may be harvested from the animal source and used as adonor ligament.

[0030] In the first step of the method of the invention, an intactligament is removed from the knee of a non-human animal. The joint whichserves as the source of the ligament should be collected from freshlykilled animals and preferably immediately placed in a suitable sterileisotonic or other tissue preserving solution. Harvesting of the jointsshould occur as soon as possible after slaughter of the animal andpreferably should be performed in the cold, i.e., in the approximaterange of about 5° C. to about 20° C., to minimize enzymatic degradationof the ligament tissue.

[0031] The ligaments are harvested from the joints in the cold, understrict sterile technique. The joint is opened by standard surgicaltechnique. Preferably, the ligament is harvested with a block of boneattached to one or both ends, although in some forms of the inventionthe ligament alone is harvested. In one form of the invention, a blockof bone representing a substantially cylindrical plug of approximately9-10 mm in diameter by 20-40 mm in length may be left attached to theligament. The ligament is carefully identified and dissected free ofadhering tissue, thereby forming the xenograft.

[0032] The xenograft is then washed in about ten volumes of sterile coldwater to remove residual blood proteins and water soluble materials. Thexenograft is then immersed in alcohol at room temperature for about fiveminutes, to sterilize the tissue and to remove non-collagenousmaterials.

[0033] After alcohol immersion, the xenograft may be directly implantedinto a knee. Alternatively, the xenograft may be subjected to at leastone of the following treatments: radiation treatment, treatment withalcohol or ozonation, one or more cycles of freezing and thawing, andtreatment with a chemical cross-linking agent. When more than one ofthese treatments is applied to the xenograft, the treatments may occurin any order.

[0034] In one embodiment of the method of the invention, the xenograftmay be treated by exposure to ultraviolet radiation for about fifteenminutes or gamma radiation in an amount of about 0.5 to 3 MegaRad.

[0035] In another embodiment, the xenograft may be treated by againbeing placed in an alcohol solution. Any alcohol solution may be used toperform this treatment. Preferably, the xenograft is placed in a 70%solution of isopropanol at room temperature.

[0036] In still another embodiment, the xenograft may be subjected toozonation.

[0037] In a further embodiment of the method of the invention, thexenograft may be treated by freeze/thaw cycling. For example, thexenograft may be frozen using any method of freezing, so long as thexenograft is completely frozen, i.e., no interior warm spots remainwhich contain unfrozen tissue. Preferably, the xenograft is dipped intoliquid nitrogen for about five minutes to perform this step of themethod. More preferably, the xenograft is frozen slowly by placing it ina freezer. In the next step of the freeze/thaw cycling treatment, thexenograft is thawed by immersion in an isotonic saline bath at roomtemperature (about 25° C.) for about ten minutes. No external heat orradiation source is used, in order to minimize fiber degradation.

[0038] In yet a further embodiment, the xenograft may optionally beexposed to a chemical agent to tan or crosslink the proteins within theextracellular proteins, to further diminish or reduce the immunogenicdeterminants present in the xenograft. Any tanning or crosslinking agentmay be used for this treatment, and more than one crosslinking step maybe performed or more than one crosslinking agent may be used in order toensure complete crosslinking and thus optimally reduce theimmunogenicity of the xenograft. For example, aldehydes such asglutaraldehyde, formaldehyde, adipic dialdehyde, and the like, may beused to crosslink the extracellular collagen in accordance with themethod of the invention. Other suitable crosslinking agents includealiphatic and aromatic diamines, carbodiimides, diisocyanates, and thelike. When glutaraldehyde is used as the crosslinking agent, forexample, the xenograft may be placed in a buffered solution containingabout 0.05 to about 5.0% glutaraldehyde and having a pH of about 7.4.Any suitable buffer may be used, such as phosphate buffered saline ortrishydroxymethylaminomethane, and the like, so long as it is possibleto maintain control over the pH of the solution for the duration of thecrosslinking reaction, which may be from one to fourteen days, andpreferably from three to five days. Alternatively, the xenograft can beexposed to a crosslinking agent in a vapor form, including, but notlimited to, a vaporized aldehyde crosslinking agent, such as, forexample, vaporized formaldehyde. The vaporized crosslinking agent canhave a concentration and a pH and the xenograft can be exposed to thevaporized crosslinking agent for a period of time suitable to permit thecrosslinking reaction to occur. For example, the xenograft can beexposed to vaporized crosslinking agent having a concentration of about0.05 to about 5.0% and a pH of about 7.4, for a period of time which canbe from one to fourteen days, and preferably from three to five days.Exposure to vaporized crosslinking agent can result in reduced residualchemicals in the xenograft from the crosslinking agent exposure. Thecrosslinking reaction should continue until the immunogenic determinantsare substantially removed from the xenogeneic tissue, but the reactionshould be terminated prior to significant alterations of the mechanicalproperties of the xenograft. When diamines are also used as crosslinkingagents, the glutaraldehyde crosslinking should occur after the diaminecrosslinking, so that any unreacted diamines are capped. After thecrosslinking reactions have proceeded to completion as described above,the xenograft should be rinsed to remove residual chemicals, and0.01-0.05 M glycine may be added to cap any unreacted aldehyde groupswhich remain.

[0039] In addition to or in lieu of the above treatments, the xenograftcan be subjected to a cellular disruption treatment to kill thexenograft's cells, which precedes or follows digestion of the xenograftwith glycosidases to remove surface carbohydrate moieties from thexenograft. The glycosidase concentration is in a range about 1 mU/ml toabout 1000 U/ml, and preferably, in the range of about 10 U/ml to about500 U/ml, and most preferably, in the range of about 100 U/ml to 200U/ml. The glycosidase digestion in turn can be followed by linkage withcapping molecules such as sialic acid to cap surface N-acetyllactosamineends of carbohydrate chains of the xenograft.

[0040] In an embodiment of this method of the invention, the xenograftis subjected to a cellular disruption treatment to kill the cells of theligament prior to in vitro digestion of the xenograft with glycosidases.Typically after surface carbohydrate moieties have been removed fromnucleated cells and extracellular components, nucleated cells, i.e.,living cells reexpress the surface carbohydrate moieties. Reexpressionof antigenic moieties of a xenograft can provoke continued immunogenicrejection of the xenograft. In contrast, non-nucleated, i.e., deadcells, are unable to reexpress surface carbohydrate moieties. Removal ofantigenic surface carbohydrate moieties from the non-nucleated cells andextracellular components of a xenograft substantially permanentlyeliminates antigenic surface carbohydrate moieties as a source ofimmunogenic rejection of the xenograft.

[0041] Accordingly, in the above-identified embodiment, the xenograft ofthe present invention is subjected to freeze/thaw cycling as discussedabove to disrupt, i.e., to kill the cells of the ligament.Alternatively, the xenograft of the present invention is treated withgamma radiation having an amount of 0.2 MegaRad up to about 3 MegaRad.Such radiation kills the ligament cells and sterilizes the xenograft.Once killed, the ligament cells are no longer able to reexpressantigenic surface carbohydrate moieties such -gal epitopes which arefactors in the immunogenic rejection of the transplanted xenografts.

[0042] Either before or after the ligament cells are killed, thexenograft is subjected to in vitro digestion of the xenograft withglycosidases, and specifically galactosidases, such as -galactosidase,to enzymatically eliminate antigenic surface carbohydrate moieties. Inparticular, -gal epitopes are eliminated by enzymatic treatment with-galactosidases, as shown in the following reaction:

[0043] The N-acetyllactosamine residues are epitopes that are normallyexpressed on human and mammalian cells and thus are not immunogenic. Thein vitro digestion of the xenograft with glycosidases is accomplished byvarious methods. For example, the xenograft can be soaked or incubatedin a buffer solution containing glycosidase. In addition, the xenograftcan be pierced to increase permeability, as further described below.Alternatively, a buffer solution containing the glycosidase can beforced under pressure into the xenograft via a pulsatile lavage process.

[0044] Elimination of the -gal epitopes from the xenograft diminishesthe immune response against the xenograft. The -gal epitope is expressedin nonprimate mammal and in New World monkeys (monkeys of South America)as 1×10⁶-35×10⁶ epitopes per cell, as well as on macromolecules such asproteoglycans of the extracellular components. U. Galili et al., Man,apes, and Old World monkeys differ from other mammals in the expressionof -galactosyl epitopes on nucleated cells, 263 J. Biol. Chem. 17755(1988). This epitope is absent in Old World primates (monkeys of Asiaand Africa and apes) and humans, however. Id. Anti-Gal is produced inhumans and primates as a result of an immune response to -gal epitopecarbohydrate structures on gastrointestinal bacteria. U. Galili et al.,Interaction between human natural anti-galactosyl immunoglobulin G andbacteria of the human flora, 56 Infect. Immun. 1730 (1988); R. M.Hamadeh et al., Human natural anti-Gal IgG regulates alternativecomplement pathway activation on bacterial surfaces, 89 J. Clin. Invest.1223 (1992). Since nonprimate mammals produce -gal epitopes,xenotransplantation of xenografts from these mammals into primatesresults in rejection because of primate anti-Gal binding to theseepitopes on the xenograft. The binding results in the destruction of thexenograft by complement fixation and by antibody dependent cellcytotoxicity. U. Galili et al., Interaction of the natural anti-Galantibody with -galactosyl epitopes: A major obstacle forxenotransplantation in humans, 14 Immunology Today 480 (1993); M.Sandrin et al., Anti-pig IgM antibodies in human serum reactpredominantly with Gal 1-3Gal epitopes, 90 Proc. Natl. Acad. Sci. USA11391 (1993); H. Good et al., Identification of carbohydrate structureswhich bind human anti-porcine antibodies: implications for discordantgrafting in man. 24 Transplant. Proc. 559 (1992); B. H. Collins et al.,Cardiac xenografts between primate species provide evidence for theimportance of the -galactosyl determinant in hyperacute rejection, 154J. Immunol. 5500 (1995). Furthermore, xenotransplantation results inmajor activation of the immune system to produce increased amounts ofhigh affinity anti-Gal. Accordingly, the substantial elimination of -galepitopes from cells and from extracellular components of the xenograft,and the prevention of reexpression of -gal epitopes can diminish theimmune response against the xenograft associated with anti-Gal antibodybinding with -gal epitopes.

[0045] Following treatment with glycosidase, the remaining carbohydratechains (e.g., glycosaminoglycans) of the xenograft are optionallytreated with capping molecules to cap at least a portion of theremaining carbohydrate chains. This capping treatment involves cappingmolecules having a concentration range of about 0.01 mM to about 100 mM,and preferably, a concentration of about 0.1 mM to about 10 mM, and mostpreferably, a concentration of about 1 mM to about 4 mM. Treatment withcapping molecules is applicable to both glycosidase-treated andnon-glycosidase-treated xenografts. For example, xenografts from knockout animals which may lack -gal epitopes may be treated with cappingmolecules to cap carbohydrate moieties on the xenograft, therebyreducing the xenograft's immunogenicity. Examples of capping moleculesused in the present invention include fucosyl, n-acetyl glucosamine andsialic acid.

[0046] In addition, selected capping molecules, such as sialic acid, arenegatively charged. The replacement of -gal epitopes with negativelycharged molecules can further diminish immunogenic rejection of thexenograft. It is theorized that the decreased immunogenicity of thexenograft results because the negative charges conferred by the cappingmolecules repel negatively charged antibody molecules and/or cells ofthe immune system, thereby masking immunogenic regions of the xenograft.

[0047] In general, electrostatic repulsion termed “zeta potential,”prevents the interaction between molecules, other than ligands and theircorresponding receptors, in the body, and serves as a barrier againstnonspecific interactions. For example, sialic acid on carbohydratechains of envelope glycoproteins helps infectious viruses to evadeeffective recognition by antibodies and by antigen presenting cells. T.W. Rademacher et al., Glycobiology, Ann. Rev. Biochem., 57:785 (1988).Bacteria such as Neisseria gonorrhea can prevent their immunedestruction by coating themselves with sialic acid using a bacterialsialyltransferase. R. F. Rest et al., Neisseria sialyltransferases andtheir role in pathogenesis, Microbial Pathogenesis, 19:379 (1995).Similarly, the protozoan Trypanosoma cruzi can infect humans and causeChagas' disease because of effective sialylation of its cell surfaceglycoproteins with sialic acid by use of the enzyme transialidase whichtransfers sialic acid from host glycoproteins to carbohydrate chains onthe parasite's membrane. O. Previato et al., Incorporation of sialicacid into Trypanosoma cruzi macromolecules, A proposalfor new metabolicroute, Mol. Biochem. Parasitol., 16:85 (1985); B. Zingales et al.,Direct sialic acid transfer from a protein donor to glycolipids oftrypomastigote forms of Trypanosoma cruzi, Mol. Biochem. Parasitol.,26:1335 (1987). Decreasing immunogenicity by sialic acid is a methodalso used by mammalian cells. Normal antigen presenting cells preventnonspecific adhesion with T lymphocytes by the expression of a highlysialylated protein named sialophorin (also termed CD43). E.Famole-Belasio et al., Antibodies against sialophorin (CD43) enhance thecapacity of dendritic cells to cluster and activate T lymphocytes., J.Immunol., 159:2203 (1997). Many malignant cell types that acquiremetastatic properties, increase the expression of sialic acid on theircell surface glycoproteins and thus mask their tumor antigens anddecrease the possibility of their detection and destruction by theimmune system. G. Yogeswarren et al., Metastatic potential is positivelycorrelated with cell surface sialylation of cultural murine cells,Science, 212:1514 (1981); J. W. Dennis, Changes in glycosylationassociated with malignant transformation and tumor progression. In: Cellsurface carbohydrates and cell development, M. Fukuda, Ed. CRC Press,pp. 161-213 (1992).

[0048] The same strategy for prevention of immune recognition can beimplemented by treatment of -galactosidase treated xenografts withnegatively charged molecules. The addition of negatively chargedmolecules to the ends of the carbohydrate chains on the cells and/or onthe extracellular molecules of the -galactosidase treated xenografts canmask the non-Gal antigens of the xenograft and diminish immunogenicrejection of the xenograft.

[0049] Sialic acid is a non-limiting example of a negatively chargedcapping molecule used to cap the carbohydrate chains of the xenograft ofthe present invention. Sialic acid can be linked in vitro to thecarbohydrate chains of the xenograft by sialyltransferase (ST),preferably in a concentration of about 1 mU/ml to about 1000 U/ml, andmore preferably in a concentration of about 10 U/ml to about 200 U/ml,in the following exemplary reaction:

[0050] Sialic acid can also be linked in vitro to the carbohydratechains of the xenograft by recombinant trans-sialidase (TS), preferablyin a concentration of about 1 mU/ml to about 1000 U/ml, and morepreferably in a concentration of about 10 U/ml to about 200 U/ml, in thefollowing exemplary reaction:

[0051] Prior to treatment, the outer surface of the xenograft mayoptionally be pierced to increase permeability to agents used to renderthe xenograft substantially non-immunogenic. A sterile surgical needlesuch as an 18 gauge needle may be used to perform this piercing step,or, alternatively a comb-like apparatus containing a plurality ofneedles may be used. The piercing may be performed with variouspatterns, and with various pierce-to-pierce spacings, in order toestablish a desired access to the interior of the xenograft. Piercingmay also be performed with a laser. In one form of the invention, one ormore straight lines of punctures about three millimeters apart areestablished circumferentially in the outer surface of the xenograft.

[0052] Prior to implantation, the ligament xenograft of the inventionmay be treated with limited digestion by proteolytic enzymes such asficin or trypsin to increase tissue flexibility or coated withanticalcification agents, antithrombotic coatings, antibiotics, growthfactors, or other drugs which may enhance the incorporation of thexenograft into the recipient knee joint. The ligament xenograft of theinvention may be further sterilized using known methods, for example,with additional glutaraldehyde or formaldehyde treatment, ethylene oxidesterilization, propylene oxide sterilization, or the like. The xenograftmay be stored frozen until required for use.

[0053] The ligament xenograft of the invention, or a segment thereof,may be implanted into a damaged human knee joint by those of skill inthe art using known arthroscopic surgical techniques. Specificinstruments for performing arthroscopic techniques are known to those ofskill in the art, which ensure accurate and reproducible placement ofligament implants. Initially, complete diagnostic arthroscopy of theknee joint is accomplished using known methods. The irreparably damagedligament is removed with a surgical shaver. The anatomic insertion sitesfor the ligament are identified and drilled to accommodate a bone plug.The size of the bone plug can be about 9-10 mm in width by about 9-10 mmin depth by 20-40 mm in length. The xenogeneic ligament is broughtthrough the drill holes and affixed with interference screws. Routineclosure is performed.

[0054] This invention is further illustrated by the following Exampleswhich should not be construed as limiting. The contents of allreferences and published patents and patent applications citedthroughout the application are hereby incorporated by reference.

EXAMPLE 1

[0055] Assay For -Gal Epitopes' Elimination From Ligament By-Galactosidase

[0056] In this example, an ELISA assay for assessing the elimination of-gal epitopes from ligament is conducted.

[0057] A monoclonal anti-Gal antibody (designated M86) which is highlyspecific for -gal epitopes on glycoproteins is produced by fusion ofsplenocytes from anti-Gal producing knock-out mice for 1,3galactosyltransferase, and a mouse hybridoma fusion partner.

[0058] The specificity of M86 for -gal epitopes on glycoproteins isillustrated in FIG. 1. M86 binds to synthetic -gal epitopes linked to•-bovine serum albumin (BSA), to

-bovine thyroglobulin which has 11 -gal epitopes, R. G. Spiro et al.,Occurrence of -D-galactosyl residues in the thyroglobulin from severalspecies. Localization in the saccharide chains of complex carbohydrates,259 J. Biol. Chem. 9858 (1984); or to ▪-mouse laminin which has 50 -galepitopes, R. G. Arumugham et al., Structure of the asparagine-linkedsugar chains of laminin. 883 Biochem. Biophys. Acta 112 (1986); but notto □-human thyroglobulin or human laminin, O-Gal 1-4 G1cNAc-BSA(N-acetyllactosamine-BSA) and Gal 1-4Gal 1-4G1cNAc-BSA (P1 antigenlinked to BSA), all of which completely lack -gal epitopes. Binding ismeasured at different dilutions of the M86 tissue culture medium.

[0059] Once the M86 antibody is isolated, the monoclonal antibody isdiluted from about 1:20 to about 1:160, and preferably diluted fromabout 1:50 to about 1:130. The antibody is incubated for a predeterminedperiod of time ranging between about 5 hr to about 24 hr, at apredetermined temperature ranging from about 3° C. to about 8° C. Theantibody is maintained in constant rotation with fragments of ligamentabout 5 m to about 100 m in size, and more preferably with ligamentfragments ranging from about 10 m to about 50 m in size, at variousligament concentrations ranging from about 200 mg/ml to about 1.5 mg/ml.Subsequently, the ligament fragments are removed by centrifugation atcentrifugation rate ranging from about 20,000×g to about 50,000×g. Theproportion of M86 bound to the ligament is assessed by measuring theremaining M86 activity in the supernatant, in ELISA with -gal-BSA asdescribed in the prior art in, for example, U. Galili et al., Porcineand bovine cartilage transplants in cynomolgus monkey: II. Changes inanti-Gal response during chronic rejection, 63 Transplantation 645-651(1997). The extent of binding of M86 to the ligament is defined as apercentage inhibition of subsequent binding to -gal-BSA. There is adirect relationship between the amount of -gal epitopes in the ligamentand the proportion of M86 complexed with the ligament fragments, thusremoved from the supernatant (i.e., percentage inhibition).Subsequently, the ligament fragments are removed by centrifugation at35,000×g and the remaining M86 in the supernatant is assessed in ELISAwith -gal-BSA as solid phase antigen.

EXAMPLE 2

[0060] Assessment Of Primate Response To Implanted Bovine LigamentTreated With -Galactosidase

[0061] In this example, bovine ligament implants are treated with-galactosidase to eliminate -galactosyl epitopes, the implants aretransplanted into cynomolgus monkeys, and the primate response to theligament implants is assessed.

[0062] A bovine stifle joint is sterilely prepared and a ligament with ablock of bone attached to one or both ends is removed in the cold, understrict sterile technique. A block of bone representing a substantiallycylindrical plug of approximately 9 mm in diameter by 40 mm in length isleft attached to the ligament. The ligament is carefully identified anddissected free of adhering tissue, thereby forming the xenograft.

[0063] The xenograft is then washed for at least five minutes with analcohol, such as ethanol or isopropanol, to remove synovial fluid andlipid soluble contaminants.

[0064] The ligament specimen is frozen at a temperature of about−70° C.to disrupt, that, is to kill, the ligament specimen's cells.

[0065] Each ligament specimen is cut into two portions. The firstportion is immersed in a buffer, such as citrate buffer solution, with apH ranging from about 5 to about 6. The buffer contains -galactosidaseat a concentration ranging from about 50 U/ml to about 300 U/ml and anadditive, such as PEG, ranging in a concentration of about 2% to about6%. The ligament/ -galactosidase buffer solution is incubated at atemperature ranging from about 25° C. to about 32° C. for apredetermined period of time ranging from about one hr to about six hr.

[0066] At the end of the incubation, the first portion is washed underconditions which allow the enzyme to diffuse out. For example, in thepresent example, the ligament is washed twice with citrate buffer andthree times with phosphate-buffered saline (PBS) pH 7.5. Each wash caninclude incubation in 50 ml of buffer solution for 10 min with gentlerocking at 24° C. Other washing procedures known to those of ordinaryskill in the art can also be used. Assays are performed to confirm thecomplete removal of the -gal epitopes.

[0067] Confirmation of complete removal of -gal epitopes is performedusing the ELISA inhibition assay with the monoclonal anti-Gal M86antibody, as described above in Example 1. The -galactosidase isproduced according to the methods known in the prior art, such as, forexample, the methods described in A. Zhu et al., Characterization ofrecombinant -galactosidase for use in seroconversion from blood group Bto O of human erythrocytes, 827 Arch. Biochem. Biophysics 324 (1996); A.Zhu et al., High-level expression and purification of coffee bean-galactosidase produced in the yeast Pichia pastoris, 827 Arch. Biochem.Biophysics 324 (1996).

[0068] Each ligament sample is implanted in the supra patellar pouch ofsix cynomolgus monkeys. With the animals under general inhalationanesthesia, the anatomic insertion sites for the xenogeneic ligament areidentified and drilled to accommodate a substantially 9 mm in diameterby 40 mm in length bone plug. The xenogeneic ligament is brought throughthe drill holes and affixed with interference screws. The procedure isperformed under sterile surgical technique, and the wounds are closedwith 3-0 vicryl or a suitable equivalent known to those of ordinaryskill in the art. The animals are permitted unrestricted cage activityand monitored for any sign of discomfort, swelling, infection, orrejection. Blood samples (e.g., 2 ml) are drawn periodically (e.g.,every two weeks) for monitoring of antibodies.

[0069] The occurrence of an immune response against the xenograft isassessed by determining anti-Gal and non-anti-Gal anti-cartilageantibodies (i.e., antibodies binding to cartilage antigens other thanthe -gal epitopes) in serum samples from the transplanted monkeys. Atleast two ml blood samples are drawn from the transplanted monkeys onthe day of implant surgery and at periodic (e.g., two week) intervalspost-transplantation. The blood samples are centrifuged and the serumsamples are frozen and evaluated for the anti-Gal and other non-anti-Galanti-cartilage antibody activity.

[0070] Anti-Gal activity is determined in the serum samples in ELISAwith -gal-BSA as solid phase antigen, according to methods known in theprior art, such as, for example, the methods described in Galili et al.,Porcine and bovine cartilage transplants in cynomolgus monkey: II.Changes in anti-Gal response during chronic rejection, 63Transplantation 645-651 (1997). For example, the -gal-BSA antigen isused to coat ELISA microtiter wells. Subsequent to blocking of the wellswith 1% BSA in PBS, sera is added to the wells in two fold serialdilutions, and incubated for 2 hr at room temperature. The plates arewashed, and incubated with secondary anti-IgG antibody conjugated toperoxidase. Color reaction is performed with o-phenylenediamine.Anti-Gal activity at the various post-transplantation serum dilutionsare compared with the baseline pretransplantation serum.

[0071] Assays are conducted to determine whether -galactosidase treatedxenografts induce the formation of anti-ligament antibodies. Formeasuring anti-ligament antibody activity, an ELISA assay is performedaccording to methods known in the prior art, such as, for example, themethods described in K. R. Stone et al., Porcine and bovine cartilagetransplants in cynomolgus monkey: I. A model for chronic xenograftrejection, 63 Transplantation 640-645 (1997). For example, a solution ofligament homogenate at 100 g/ml in carbonate buffer is used as solidphase antigen. Other buffers known to those of ordinary skill in the artcan also be used. Approximately 5 g of ligament antigens per well aredried and the wells are blocked with BSA. The serum samples used forthis assay are depleted of anti-Gal by adsorption on rabbit red cellsfor 30 min at 4° C. (at 3:1 ration vol/vol). Under these conditions, allanti-Gal antibodies are adsorbed on the many -gal epitopes expressed onrabbit red cells. U. Galili et al., Evolutionary relationship betweenthe anti-Gal antibody and the Gal 163Gal epitope in primates, 84 Proc.Natl. Acad. Sci. (USA) 1369 (1987); U. Galili et al., Contribution ofanti-Gal to primate and human IgG binding to porcine endothelial cells,60 Transplantation 210 (1995). The adsorbed sera at various dilutionsare analyzed for anti-ligament antibodies by ELISA, and thepost-transplantation production of such antibodies are assessed bycomparing this antibody activity with that observed in thepretransplantation serum.

[0072] The ligament xenograft is optionally explanted at one to twomonths post-transplantation, sectioned and stained for histologicalevaluation of inflammatory infiltrates. Post-transplantation changes inanti-Gal and other anti-ligament antibody activities are correlated withthe inflammatory histologic characteristics (i.e., granulocytes ormononuclear cell infiltrates) within the explanted ligament, one to twomonths post-transplantation, using methods known in the art, as, forexample, the methods described in K. R. Stone et al., Porcine and bovinecartilage transplants in cynomolgus monkey: I. A model for chronicxenograft rejection, 63 Transplantation 640-645 (1997).

[0073] Where the xenogeneic ligament is explanted, the ligamentxenograft is aseptically harvested. At the time of the xenograftremoval, joint fluid, if present in amounts sufficient to aspirate, iscollected from the stifle joints for possible immunologic testing if thegross and histopathologic evaluation of the transplants indicate goodperformance of the transplanted ligament.

[0074] A portion of the implant and surrounding tissue is frozen in anembedding medium for frozen tissue specimens in embedding molds forimmunohistochemistry evaluation according to the methods known in theprior art. “TISSUE-TEK®” O.C.T. compound which includes 10.24% w/wpolyvinyl alcohol, 4.26% w/w polyethylene glycol, and 86.60% w/wnonreactive ingredients, and is manufactured by Sakura FinTek, Torrence,Calif., is a non-limiting example of a possible embedding medium for usewith the present invention. Other embedding mediums known to those ofordinary skill in the art may also be used. The remaining implant andsurrounding tissue is collected in 10% neutral buffered formalin forhistopathologic examination.

EXAMPLE 3

[0075] Assessment of Primate Response to Implanted Ligament Treated with-Galactosidase, Sialic Acid-Cytosine Monophosphate and Sialyltransferase

[0076] In this example, bovine ligament implants are treated with-galactosidase to eliminate -gal epitopes, as described in Example 2.The implants are further treated with sialic acid-cytosine monophosphate(SA-CMP) and sialyltransferase to cap carbohydrate chains with sialicacid. Sialytransferase facilitates the transfer of the sialic acid fromthe SA-CMP compound to the xenograft. The sialic acid links to and thuscaps the carbohydrate chains. The cytosine monophosphate provides thenecessary energetic level to the sialic acid for such linking andcapping. Capping with sialic acid interferes with the ability of the thesubject's immune system to recognize the xenograft as foreign. Thenegative charge of the sialic acid further interferes with the abilityof the ligament antigens to bind with non-anti-Gal anti-cartilageantibodies (i.e., antibodies binding to ligament antigens other than the-gal epitopes.) The implants are transplanted into cynomolgus monkeys,and the primate response to the ligament implants is assessed.

[0077] Bovine ligament stifle joints are prepared as described inExample 2 including the -galactosidase treatment. Prior to implantationinto the monkeys, however, the implants are further treated with apredetermined amount of a predetermined amount of SA-CMP andsialyltransferase, at specified concentrations for a predetermined timeand at a predetermined temperature, to cap carbohydrate chains withsialic acid. For example, the sample is immersed in 10 ml buffersolution at a pH of about 5.5 to 7.0, and preferably a pH of about6.0-6.5, and most preferably a pH of about 6.2, containing SA-CMP at aconcentration of approximately about 1 mM to about 10 mM, andsialyltransferase at a concentration of about 100 U/ml. The sample isincubated at a temperature range of about 26° C. to about 37° C. for apredetermined time period of about one hr to about four hr.

[0078] Other enzymes such as recombinant transialidase can be used tofacilitate the transfer of sialic acid from compounds such as sialylatedlactose to the xenograft.

[0079] Further, other molecules, such as fucosyl in combination with thecorresponding fucosyltransferase and n-acetyl glucosamine in combinationwith the corresponding glycosyltransferase, can also be used for cappingthe carbohydrate chains of the implants.

[0080] Subsequently, the samples are washed to remove the enzyme andimplanted into the monkeys, and the occurrence of an immune responseagainst the xenograft is assessed as described above in Example 2.

[0081] Those of skill in the art will recognize that the invention maybe embodied in other specific forms without departing from the spirit oressential characteristics thereof. The presently described embodimentsare therefore to be considered in all respects as illustrative and notrestrictive, the scope of the invention being indicated by the appendedclaims rather than by the foregoing description, and all variations ofthe invention which are encompassed within the meaning and range ofequivalency of the claims are therefor intended to be embraced therein.

What is claimed is:
 1. A method of preparing a ligament xenograft forimplantation into a human, which comprises: (a) removing at least aportion of a ligament from a non-human animal to provide a xenograft;(b) washing the xenograft in water and alcohol; (c) subjecting thexenograft to a cellular disruption treatment; and (d) digesting thexenograft with a glycosidase to remove substantially a plurality offirst surface carbohydrate moieties from the xenograft, wherein theglycosidase has a concentration in a range of about 1 mU/ml to about1000 U/ml, and whereby the xenograft has substantially the samemechanical properties as a corresponding portion of a native ligament.2. The method of claim 1, further comprising the step of: subsequent tothe glycosidase digesting step, treating a plurality of second surfacecarbohydrate moieties on the xenograft with a plurality of cappingmolecules to cap at least a portion of the second surface carbohydratemoieties, whereby the xenograft is substantially non-immunogenic.
 3. Themethod of claim 2, wherein the capping step comprises: treating thesecond surface carbohydrate moieties on the xenograft with the cappingmolecules having a concentration in a range of about 0.01 mM to about100 mM.
 4. The method of claim 2, wherein at least a portion of thecapping molecules are sialic acid molecules.
 5. The method of claim 1,wherein the glycosidase is a galactosidase.
 6. The method of claim 5,wherein the galactosidase is an -galactosidase.
 7. The method of claim1, wherein the cellular disruption treatment comprises freeze/thawcycling.
 8. The method of claim 1, wherein the cellular disruptiontreatment comprises exposure to gamma radiation.
 9. The method of claim1, wherein the removing step comprises removing with the portion a firstblock of bone attached to a first end of the portion.
 10. The method ofclaim 9, wherein the removing step comprises removing with the portion asecond block of bone affixed to a second end of the portion opposite thefirst end.
 11. The method of claim 1 further comprising the step offollowing step (c), exposing the xenograft to a crosslinking agent in avapor form.
 12. A method of preparing a meniscal xenograft forimplantation into a human, which comprises: (a) removing at least aportion of a ligament from a non-human animal to provide a xenograft;(b) washing the xenograft in water and alcohol; (c) subjecting thexenograft to a cellular disruption treatment; (d) digesting thexenograft with a glycosidase to remove substantially a plurality offirst surface carbohydrate moieties from the xenograft; and (e) treatinga plurality of second surface carbohydrate moieties on the xenograftwith a plurality of sialic acid molecules to cap at least a portion ofthe second surface carbohydrate moieties, whereby the xenograft issubstantially non-immunogenic and has substantially the same mechanicalproperties as a corresponding portion of a native ligament.
 13. Themethod of claim 12, wherein the capping step comprises: treating thesecond surface carbohydrate moieties on the xenograft with the sialicacid molecules having a concentration in a range of about 0.01 mM toabout 100 mM.
 14. The method of claim 12, wherein at least theglycosidase is a galactosidase.
 15. The method of claim 14, wherein atleast the galactosidase is an -galactosidase.
 16. The method of claim12, wherein the cellular disruption treatment comprises freeze/thawcycling.
 17. The method of claim 12, wherein the cellular disruptiontreatment comprises exposure to gamma radiation.
 18. The method of claim12, wherein the removing step comprises removing with the portion afirst block of bone attached to a first end of the portion.
 19. Themethod of claim 18, wherein the removing step comprises removing withthe portion a second block of bone affixed to a second end of theportion opposite the first end.
 20. The method of claim 12 furthercomprising the step of: following step (c), exposing the xenograft to acrosslinking agent in a vapor form.
 21. An article of manufacturecomprising a substantially non-immunogenic ligament xenograft forimplantation in to a human, produced by (a) removing at least a portionof a ligament from a non-human animal to provide a xenograft; (b)washing the xenograft in water and alcohol; (c) subjecting the xenograftto a cellular disruption treatment; and (d) digesting the xenograft witha glycosidase to remove substantially a plurality of first surfacecarbohydrate moieties from the xenograft, wherein the glycosidase has aconcentration in a range of about 1 mU/ml to about 1000 U/ml, andwhereby the xenograft has substantially the same mechanical propertiesas a corresponding portion of a native ligament.
 22. The article ofmanufacture of claim 21, further produced by: subsequent to theglycosidase digesting step, treating a plurality of second surfacecarbohydrate moieties on the xenograft with a plurality of cappingmolecules to cap at least a portion of the second surface carbohydratemoieties on the xenograft, whereby the xenograft is substantiallynon-immunogenic.
 23. The article of manufacture of claim 22, wherein thecapping molecules have a concentration in a range of about 0.01 mM toabout 100 mM.
 24. The article of manufacture of claim 22, wherein atleast a portion of the capping molecules are sialic acid molecules. 25.The article of manufacture of claim 21, wherein the glycosidase is agalactosidase.
 26. The article of manufacture of claim 25, wherein thegalactosidase is an -galactosidase.
 27. The article of manufacture ofclaim 21, wherein the cellular disruption treatment comprisesfreeze/thaw cycling.
 28. The article of manufacture of claim 21, whereinthe cellular disruption treatment comprises exposure to gamma radiation.29. The article of manufacture of claim 21, wherein the removing stepcomprises removing with the portion a first block of bone attached to afirst end of the portion.
 30. The article of manufacture of claim 29,wherein the removing step comprises removing with the portion a secondblock of bone affixed to a second end of the portion opposite the firstend.
 31. The article of manufacture of claim 21 further comprising thestep of following step (c), exposing the xenograft to a crosslinkingagent in a vapor form.
 32. An article of manufacture comprising asubstantially non-immunogenic ligament xenograft for implantation in toa human, produced by: (a) removing at least a portion of a ligament froma non-human animal to provide a xenograft; (b) washing the xenograft inwater and alcohol; (c) subjecting the xenograft to a cellular disruptiontreatment; (d) digesting the xenograft with a glycosidase to removesubstantially a plurality of first surface carbohydrate moieties fromthe xenograft; and (e) treating a plurality of second surfacecarbohydrate moieties on the xenograft with a plurality of sialic acidmolecules to cap at least a portion of the second surface carbohydratemoieties, whereby the xenograft is substantially non-immunogenic and hassubstantially the same mechanical properties as a corresponding portionof a native meniscus.
 33. The article of manufacture of claim 32,wherein the sialic acid molecules have a concentration in a range ofabout 0.01 mM to about 100 mM.
 34. The article of manufacture of claim32, wherein the glycosidase is a galactosidase.
 35. The article ofmanufacture of claim 34, wherein the galactosidase is an -galactosidase.36. The article of manufacture of claim 32, wherein the cellulardisruption treatment comprises freeze/thaw cycling.
 37. The article ofmanufacture of claim 32, wherein the cellular disruption treatmentcomprises exposure to gamma radiation.
 38. The article of manufacture ofclaim 32, wherein the removing step comprises removing with the portiona first block of bone attached to a first end of the portion.
 39. Thearticle of manufacture of claim 38, wherein the removing step comprisesremoving with the portion a second block of bone affixed to a second endof the portion opposite the first end.
 40. The article of manufacture ofclaim 32 further comprising the step of: following step (c), exposingthe xenograft to a crosslinking agent in a vapor form.
 41. A ligamentxenograft for implantation into a human comprising: a portion of aligament from a non-human animal, wherein the portion includes aplurality of extracellular components and a plurality of substantiallyonly dead cells, the extracellular components and the dead cells havingsubstantially no surface -galactosyl moieties and having a plurality ofsialic acid molecules linked to at least a portion of a plurality ofsurface carbohydrate moieties on the xenograft, whereby the portion ofthe ligament is substantially non-immunogenic and has substantially thesame mechanical properties as a corresponding portion of a nativeligament.
 42. The ligament xenograft of claim 41, wherein the portion ofthe ligament has a first block of bone attached to a first end thereof.43. The ligament xenograft of claim 42, wherein the portion of theligament has a second block of bone affixed to a second end thereofopposite the first end.